Ca2+ ions function as intracellular second messengers that regulate a wide variety of cellular activities including fertilization, apoptosis, muscle contraction, lymphocyte activation, and, most pertinent to this proposal, cell cycle control and proliferation. Precise control of the cell cycle is critical throughout development, and Ca2+ functions in signal transduction pathways that regulate the cell cycle as early as fertilization. Therefore, delineation of the Ca2+ signaling mechanisms that regulate the cell cycle is paramount to our understanding of developmental processes. A clear understanding of the basic mechanisms that regulate the cell cycle is also fundamental to our ability to prevent and treat cancer, which is caused by abnormal cell cycle regulation. The long-range goals of this project are to understand the basic mechanisms by which Ca2+ signaling regulates the cell cycle and proliferation, particularly during development, and to identify Ca2+ regulatory mechanisms that may be potential therapeutic targets in the prevention and treatment of cancer. We are only now beginning to appreciate the important role that Ca2+ signaling pathways and specific Ca2+ channels play in the development and progression of cancer, and this is a topic that requires significantly more research. A near-ubiquitous signal transduction pathway that influences the intracellular Ca2+ concentration and is likely to be involved in regulation of the cell cycle is the store-operated Ca2+ entry (SOCE) pathway. SOCE refers to Ca2+ influx that occurs as a result of depletion of endoplasmic reticulum (ER) Ca2+ stores. Ca2+ influx that occurs as a result of the SOCE pathway can be characterized electrophysiologically, and the measurable current associated with SOCE is referred to as Ca2+ release-activated Ca2+ current (Icrac). Recent breakthroughs have identified the requirements for Stim and Orai proteins in the SOCE pathway, and these seminal discoveries have facilitated molecular analysis of SOCE signaling and of its physiological targets. It has been demonstrated that in response to ER Ca2+ store depletion, Stim1 rearranges within the ER into punctuate structures near the plasma membrane where it activates Orai SOCE channels. This proposal will use molecular techniques to define the roles of Stim- and Orai-mediated SOCE during interphase and mitotic stages of the cell cycle. We hypothesize that SOCE positively regulates interphase stages of the cell cycle, but is potentially detrimental during mitosis and is therefore suppressed at this stage. The following Specific Aims will address this hypothesis: Specific Aim 1: Define the Ca2+ influx mechanisms required for interphase progression. Specific Aim 2: Define the mechanism of SOCE suppression in mitosis. Specific Aim 3: Define the mechanisms by which the cytoplasmic Ca2+ concentration is regulated during mitosis.